The pathological diagnosis is an essential guide to prognosis, treatment and outcome of a disease; but the information is often incomplete or inexact, particularly when it is relies on histology alone. Excitingly, we are now at a moment where molecular biology is starting to change our understanding of disease, by bringing the level of our understanding to single cells, with cancer biology leading the way. Historically, kidney pathology has been particularly challenging because of the difficulty in accessing sufficient tissue. As a result, many renal diseases remain poorly characterised. We now have the opportunity to change this situation with molecular approaches that investigate renal disease at the level of single cells.
The autoimmune disease Systemic Lupus Erythematosus (SLE) is an important cause of renal disease and is a good starting point for developing our new approach to renal pathology. SLE is a chronic multisystem disorder with a complex aetiology and a strong genetic component, which is characterised by pathogenic antibodies to nuclear self-antigens. Kidney disease (lupus nephritis) occurs in about 50% of people with SLE and is a major cause of mortality and morbidity. Deposition of autoantibodies and their antigens in the renal glomeruli is necessary but not sufficient to drive tissue inflammation and renal damage. Therefore, in order to predict which patients are at risk of renal lupus and develop more effective targeted treatments, we need to understand: (i) the interplay between cells of the kidney and the immune system, and (ii) how intrarenal cellular responses, by endothelial cells, podocytes and resident renal mononuclear cells for example, play a role in determining the extent and nature of renal injury.
The project will use a mouse model of SLE, which is induced by the Toll like receptor 7 (TLR7) agonist Imiquimod, to study the renal disease and to develop new approaches to diagnosis. Intracellular TLR7 recognises viral single stranded RNA as part of innate host defences, but it can also sense endogenous ribonucleoproteins for the activation of antigen presenting cells including B cells and dendritic cells, thus contributing to autoreactive B cell proliferation and autoantibody production in SLE.
The first year will be spent characterising the TLR7-induced model, both the systemic immune phenotype and the renal cellular pathology in detail, using techniques including mass cytometry, and single cell RNA sequencing of isolated glomerular cells. The insights from this profiling will then inform further experiments to modulate the highlighted pathways, using genetic and molecular approaches, with the goal of establishing causal relationships and cell intrinsic functions, and attenuating the renal injury.
Insights into the mechanism of disease will come from work using immortalised podocyte cell lines, primary cells, and the histological, transcriptomic and proteomic study of cells and tissues ex vivo. The application of some of these methods to renal glomeruli is novel and will require creative strategies, but has a potential for translation to the analysis of SLE and other renal disease in human tissue and cells. If time allows, the same molecular pathology approaches will be applied to other models renal disease, with the eventual aim of applying them to human disease samples.
The student will be well supported to gain experience in a wide variety of molecular laboratory techniques to study renal and immunological disease. Within the Bull and Cornall labs there is significant experience with cutting edge genomics and imaging resources available within the Wellcome Centre for Human Genetics, including flow cytometry, FRET-FLIM, RNA sequencing and CRISPR/Cas9 gene editing. Further strong support and access to technology and resources will come from our links with the Weatherall Institute for Molecular Medicine, the Nuffield Dept. of Medicine and the wider University community.
This programme is suitable for someone with a background in Biomedical Science, Genetics, Immunology, Medicine or similar degree who is interested in using a wide range of cellular, biochemical and genetic approaches to study autoimmunity in the kidney, with an emphasis on translational work with relevance for human renal disease.
The project will offer the opportunity to become skilled in a wide range of established and innovative techniques and tools, and to develop as an expert in renal pathology, cell biology and immunology. Previous specific experience of immunology or nephrology is not a requirement as training and teaching will be provided in the lab. Additionally the student will be enrolled on the MRC WIMM DPhil training course in year 1 and have access to a comprehensive range of courses in generic skills and researcher development available through the Medical Sciences Division’s skills training programme.
Prospective applicants are encouraged to contact Dr Katherine Bull (firstname.lastname@example.org) for further information.
Project reference number: 1009
|Dr Katherine Bull||Centre for Cellular and Molecular Physiology||Oxford University, Henry Wellcome Building for Molecular Physiology||GBRemail@example.com|
|Professor Richard J Cornall FMedSci FRCP||Centre for Cellular and Molecular Physiology||Oxford University, Henry Wellcome Building for Molecular Physiology||GBRfirstname.lastname@example.org|
The study of mutations causing the steroid-resistant nephrotic syndrome in children has greatly advanced our understanding of the kidney filtration barrier. In particular, these genetic variants have illuminated the roles of the podocyte, glomerular basement membrane and endothelial cell in glomerular filtration. However, in a significant number of familial and early onset cases, an underlying mutation cannot be identified, indicating that there are likely to be multiple unknown genes with roles in glomerular permeability. We now show how the combination of N-ethyl-N-nitrosourea mutagenesis and next-generation sequencing could be used to identify the range of mutations affecting these pathways. Using this approach, we isolated a novel mouse strain with a viable nephrotic phenotype and used whole-genome sequencing to isolate a causative hypomorphic mutation in Lamb2. This discovery generated a model for one part of the spectrum of human Pierson's syndrome and provides a powerful proof of principle for accelerating gene discovery and improving our understanding of inherited forms of renal disease. Hide abstract
Recent evidence suggests that systemic autoimmune disease depends on signals from TLR ligands, but little is known about how TLR-dependent pathways lead to the loss of self tolerance in vivo. To address this, we have examined the role of TLR signaling in Lyn-deficient mice, which develop an autoimmune disease similar to SLE. We found that absence of the TLR signaling adaptor molecule MyD88 suppresses plasma cell differentiation of switched and unswitched B cells, and prevents the generation of antinuclear IgG antibodies and glomerulonephritis. In mixed chimeras the increased IgM and IgG antibody secretion in Lyn-deficient mice is at least partially due to B cell-independent effects of Lyn. We now show that MyD88 deficiency blocks the expansion and activation of DC in which Lyn is also normally expressed, and prevents the hypersecretion of proinflammatory cytokines IL-6 and IL-12 by Lyn-deficient DC. These findings further highlight the important role of TLR-dependent signals in both lymphocyte activation and autoimmune pathogenesis. Hide abstract
Spontaneous germinal center (Spt-GC) B cells and follicular helper T cells generate high-affinity autoantibodies that are involved in the development of systemic lupus erythematosus. TLRs play a pivotal role in systemic lupus erythematosus pathogenesis. Although previous studies focused on the B cell-intrinsic role of TLR-MyD88 signaling on immune activation, autoantibody repertoire, and systemic inflammation, the mechanisms by which TLRs control the formation of Spt-GCs remain unclear. Using nonautoimmune C57BL/6 (B6) mice deficient in MyD88, TLR2, TLR3, TLR4, TLR7, or TLR9, we identified B cell-intrinsic TLR7 signaling as a prerequisite to Spt-GC formation without the confounding effects of autoimmune susceptibility genes and the overexpression of TLRs. TLR7 deficiency also rendered autoimmune B6.Sle1b mice unable to form Spt-GCs, leading to markedly decreased autoantibodies. Conversely, B6.yaa and B6.Sle1b.yaa mice expressing an extra copy of TLR7 and B6.Sle1b mice treated with a TLR7 agonist had increased Spt-GCs and follicular helper T cells. Further, TLR7/MyD88 deficiency led to compromised B cell proliferation and survival after B cell stimulation both in vitro and in vivo. In contrast, TLR9 inhibited Spt-GC development. Our findings demonstrate an absolute requirement for TLR7 and a negative regulatory function for TLR9 in Spt-GC formation under nonautoimmune and autoimmune conditions. Our data suggest that, under nonautoimmune conditions, Spt-GCs initiated by TLR7 produce protective Abs. However, in the presence of autoimmune susceptibility genes, TLR7-dependent Spt-GCs produce pathogenic autoantibodies. Thus, a single copy of TLR7 in B cells is the minimal requirement for breaking the GC-tolerance checkpoint. Hide abstract
Our understanding of kidney disease pathogenesis is limited by an incomplete molecular characterization of the cell types responsible for the organ's multiple homeostatic functions. To help fill this knowledge gap, we characterized 57,979 cells from healthy mouse kidneys by using unbiased single-cell RNA sequencing. On the basis of gene expression patterns, we infer that inherited kidney diseases that arise from distinct genetic mutations but share the same phenotypic manifestation originate from the same differentiated cell type. We also found that the collecting duct in kidneys of adult mice generates a spectrum of cell types through a newly identified transitional cell. Computational cell trajectory analysis and in vivo lineage tracing revealed that intercalated cells and principal cells undergo transitions mediated by the Notch signaling pathway. In mouse and human kidney disease, these transitions were shifted toward a principal cell fate and were associated with metabolic acidosis. Hide abstract
OBJECTIVE: To examine whether topical treatment of wild-type mice with Toll-like receptor 7 (TLR-7) agonists leads to lupus-like autoimmunity. METHODS: Wild-type FVB/N, BALB/c, and C57BL/6 mice were treated with the topical TLR-7 agonist imiquimod or R848 administered to the ear 3 times weekly. During treatment, the mice were monitored for serum autoantibody and creatinine levels as well as histopathology of the kidneys, spleens, livers, hearts, and skin. Immunologic abnormalities were analyzed by immunohistochemistry, quantitative reverse transcription-polymerase chain reaction, and fluorescence-activated cell sorting. The role of plasmacytoid dendritic cells (PDCs) in the development of autoimmune disease was validated by in vivo treatment with an anti-PDC antibody. Diseased mice underwent ultraviolet B irradiation, to evaluate skin photosensitivity. The disease-causing effect of topical application of imiquimod was compared with that of systemic (intraperitoneal) administration. TLR-7- and TLR-9-deficient mice were used to validate the role of TLR-7. RESULTS: Wild-type mice of different genetic backgrounds developed systemic autoimmune disease following 4 weeks of topical treatment with imiquimod or R848, with elevated levels of autoantibodies to double-stranded DNA and multiple organ involvement, including glomerulonephritis, hepatitis, carditis, and photosensitivity. Expression of Ifna and Mx1, the interferon-α-stimulated gene, was up-regulated in the organs of imiquimod-treated mice. However, disease caused by intraperitoneal injection of imiquimod was less severe than that induced by topical application. In vivo depletion of PDCs by a specific antibody protected mice against the autoimmunity induced by topical administration of imiquimod, suggesting a role of PDCs. Furthermore, TLR-7-deficient mice, but not TLR-9-deficient mice, were protected against autoimmunity. CONCLUSION: This protocol provides a novel model of inducible systemic lupus erythematosus in wild-type mice and underscores the skin as the primary organ that allows TLR-7 agonists to induce SLE. Hide abstract
Podocytes are essential to the structure and function of the glomerular filtration barrier; however, they also exhibit increased expression of MHC class II molecules under inflammatory conditions, and they remove Ig and immune complexes from the glomerular basement membrane (GBM). This finding suggests that podocytes may act as antigen-presenting cells, taking up and processing antigens to initiate specific T cell responses, similar to professional hematopoietic cells such as dendritic cells or macrophages. Here, MHC-antigen complexes expressed exclusively on podocytes of transgenic mice were sufficient to activate CD8+ T cells in vivo. In addition, deleting MHC class II exclusively on podocytes prevented the induction of experimental anti-GBM nephritis. Podocytes ingested soluble and particulate antigens, activated CD4+ T cells, and crosspresented exogenous antigen on MHC class I molecules to CD8+ T cells. In conclusion, podocytes participate in the antigen-specific activation of adaptive immune responses, providing a potential target for immunotherapies of inflammatory kidney diseases and transplant rejection. Hide abstract
OBJECTIVE: Development of proteinuria in lupus nephritis (LN) is associated with podocyte dysfunction. The NLRP3 inflammasome has been implicated in the pathogenesis of LN. The purpose of this study was to investigate whether NLRP3 inflammasome activation is involved in the development of podocyte injury in LN. METHODS: A fluorescence-labeled caspase 1 inhibitor probe was used to detect the activation of NLRP3 inflammasomes in podocytes derived from lupus-prone NZM2328 mice and from renal biopsy tissues obtained from patients with LN. MCC950, a selective inhibitor of NLRP3, was used to treat NZM2328 mice. Proteinuria, podocyte ultrastructure, and renal pathology were evaluated. In vitro, sera from diseased NZM2328 mice were used to stimulate a podocyte cell line, and the cells were analyzed by flow cytometry. RESULTS: NLRP3 inflammasomes were activated in podocytes from lupus-prone mice and from patients with LN. Inhibition of NLRP3 with MCC950 ameliorated proteinuria, renal histologic lesions, and podocyte foot process effacement in lupus-prone mice. In vitro, sera from diseased NZM2328 mice activated NLRP3 inflammasomes in the podocyte cell line through the production of reactive oxygen species. CONCLUSION: NLRP3 inflammasomes were activated in podocytes from lupus-prone mice and from LN patients. Activation of NLRP3 is involved in the pathogenesis of podocyte injuries and the development of proteinuria in LN. Hide abstract